The primary RNA transcript of the calcitonin-(CT) gene is processed by a unique splicing mechanism called "alternative splicing" to produce a messenger RNA (mRNA) coding for calcitonin or a mRNA coding for a newly described protein called calcitonin gene-related peptide (CGRP). We have demonstrated production of both of these mRNA species by the TT cells, a human medullary thyroid carcinoma cell line and have demonstrated an effect of glucocorticoids on the alternate splicing process resulting in an increase in the production of CT mRNA and a decrease in the production of CGRP mRNA. In an attempt to further understand the alternate splicing process and how it is regulated by glucocorticoids, an in vitro splicing system will be established utilizing a human genomic clone for the CT gene. Specific sequences of importance in the alter- native splicing process will be cloned into an SP6 or other transcription vector. Newly synthesized RNA from these transcription vectors will be used as substrate for an in vitro splicing assay. Questions to be asked with this system include: 1. Will nuclear extracts from glucocorticoid-treated cells splice CT RNA differently than extracts from control cells? 2. Will alteration of splice site sequences or polyadenylation site sequences change the alternative splicing process? 3. Can the specific sequences shown to affect the splicing process be used as a probe to isolate and purify factors important in the regulation of splicing? If efforts to develop an in vitro splicing system are unsuccessful, a transient transfection system to study RNA processing in intact cells will be developed. The questions to be asked in the transfection model are similar to those which could be asked in the in vitro splicing system, although it may be more difficult to use the system to purify potential splicing factors. There are now over 40 examples of genes whose RNA undergoes alternative splicing. An understanding of the splicing mechanism and how it is regulated will provide information of fundamental importance for the control of the growing number of genes processed by alternative splicing.